Alterations in nucleotide sequences can have profound effects on cells. For example, many tumors and many genetic diseases result from alteration, or mutation, of particular nucleotide sequences. Mutations in nucleotide sequences that encode proteins can result in production of proteins with altered polypeptide sequences and, in some instances, altered biological activities. Changes in the activity of a single protein can sometimes have profound effects on the physiology of an entire organism.
In order to develop effective preventive, diagnostic and therapeutic methods for treatment of cancer and hereditary diseases, we must first identify the genetic mutations that contribute to disease development. Typically, mutations are identified in studies of cloned genes whose normal sequences are already known (see, for example, Suzanne et al., Science 244:217, 1989; Kerem et al., Science 245:1073, 1989). That is, a gene is first identified as being associated with a disorder, and particular sequence changes that correlate with the diseased state are subsequently identified.
A variety of techniques have been used to identify sequence variations in nucleic acids. For example, Restriction Fragment Length Polymorphism (RFLP) analysis detects restriction sites generated by mutations or alterations in nucleotide sequences (see Kan et al., Lancet ii:910, 1978); Denaturing Gradient Gel Electrophoresis and Single Stranded DNA Electrophoretic Mobility Studies identify nucleotide sequence differences through alterations in the mobility of bands in electrophoresis gels (see Myers et al., Nature 313:495, 1985; Orita et al., Proc. Natl. Acad. Sci. USA 86:2766, 1989); Chemical Cleavage analysis identifies mismatched sites in heteroduplex DNA (see Cotton, Proc. Natl. Acad. Sci. USA 85:4397, 1988); and RNase Cleavage analysis identifies mismatched sites in RNA-DNA or RNA-RNA heteroduplexes (see Myers et al., Science 230:1242, 1985; Maniatis et al. U.S. Pat. No. 4,946,773).
A significant problem with each of the above-described methods for identifying nucleic acid sequence differences is that prior knowledge of the gene of interest is generally required. Additionally, each of these methods utilizes DNA display techniques that limit the number of different genes that can be analyzed at one time. There is a need for development of techniques that allow identification of sequence alterations and mutations without prior knowledge of which gene has been disrupted. There is also a need for the development of techniques that allow analysis of multiple different sequence alterations at the same time.